title: Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells.
abstract: Recently, we developed an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), which generates a detectable fluorescent signal in living cells that express the target RNA. Here, we show that RBMBs can also be used to image single RNA transcripts in living cells, when the target RNA is engineered to contain as few as four hybridization sites. Moreover, comparison with single-molecule fluorescence in situ hybridization confirmed that RBMBs could be used to accurately quantify the number of RNA transcripts within individual cells. Measurements of gene expression could be acquired within 30 min and using a wide range of RBMB concentrations. The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines. Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells. We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.
authors.affiliation: Department of Bioengineering, University of Pennsylvania, 210 S. 33rd Street, 240 Skirkanich Hall, Philadelphia, PA 19104, USA, Department of Biology, University of Pennsylvania, 433 S. University Ave, 102 Leidy Laboratories, Philadelphia, PA 19104, USA and Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 52241, USA.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zhang
authors.forename: Yang
authors.initials: Y
authors.lastname: Song
authors.forename: Akash Y
authors.initials: AY
authors.lastname: Shah
authors.forename: Virzhiniya
authors.initials: V
authors.lastname: Lekova
authors.forename: Arjun
authors.initials: A
authors.lastname: Raj
authors.forename: Ling
authors.initials: L
authors.lastname: Huang
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
authors.forename: Andrew
authors.initials: A
authors.lastname: Tsourkas
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019295:Computational Biology / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005354:Fibrosarcoma / Q000737:chemistry; D005453:Fluorescence; D020869:Gene Expression Profiling / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification*; D020080:Tandem Repeat Sequences; D014158:Transcription, Genetic
chemical_list: D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins